Simple rules govern the diversity of bacterial nicotianamine-like metallophores

Abstract

3In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores 1 4 mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and 1 5 yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis 1 6respectively. These metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), 1 7 CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α -ketoglutarate), 1 8 pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH), depending on the species. 1 9Here, we explored substrate specificity of CntM by combining bioinformatics and structural analysis 2 0 with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity was mainly due to 2 1 the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH 2 2 when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over 2 3 xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity towards α -2 4 ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an 2 5 aspartate at this position ensures selectivity towards pyruvate whereas an alanine leads to the 2 6 consumption of both pyruvate and α -ketoglutarate. Modifying this residue in P. aeruginosa led to a 2 7complete reversal of selectivity. Thus, opine-type metallophore diversity is mainly mediated by the 2 8absence/presence of a cntK gene encoding a histidine racemase, and the presence of an 2 9 aspartate/alanine at position 150 of CntM. These two simple rules predict the production of a fourth 3 0 metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline. 3 1 3 2 3 3 3 5metals required for their growth. This is particularly the case for pathogenic bacteria that have to 3 6 confront the host's immune system. Indeed, the so-called "nutritional immunity" induces an additional 3 7 metal limitation by sequestering iron, zinc or manganese to prevent bacterial growth [1-4]. To face 3 8 this metal restriction, bacteria have developed metallophores to recover metals. In this context, 3 9 nicotianamine-like metallophores have been identified in some bacteria as playing an important role in 4 0 metal acquisition strategies. Staphylopine, pseudopaline and yersinopine are the three examples 4 1 2 currently known and recently identified in S. aureus [5], P. aeruginosa [6,7] and Y. pestis [8] 4 2respectively. The biosynthesis of these nicotianamine-like metallophores occurs in two or three steps 4 3 depending on the species. When it is present, as in S. aureus, CntK, a histidine racemase, transforms 4 4

Structural insights into the ligand recognition and catalysis of the key aminobutanoyltransferase CntL in staphylopine biosynthesis

“…In addition to the CntLs of S. aureus and Paenibacillus mucilaginosus , which use D‐His as a substrate, recent studies identified another class of enzymes from Pseudomonas aeruginosa ( Pa NAS) and Yersinia pestis ( Yp NAS), which stereoselectively catalyze L‐His 18‐19 . Sequence alignments of these CntL/NAS enzymes revealed that 4 residues (V80, S195, T197, and F255 in Sa CntL) of the 11 residues within 5 Å of the substrate histidine are conserved within each class but different across two classes (Figure 5A).…”

“…Zn 2+ was previously considered to be mainly imported directly through canonical ABC importer systems: ZnuABC in gram‐positive bacteria, such as Pseudomonas aeruginosa ( P. aeruginosa ), 10 and AdcABC in gram‐positive bacteria, such as Staphylococcus aureus ( S. aureus ) 11‐12 . Recently, a metallophore‐based system was identified in both gram‐positive (named CntA‐F in S. aureus ) 13 and gram‐negative bacteria (named zrmABCD in P. aeruginosa ), 10,14‐15 which synthesizes a nicotianamine‐like metallophore to facilitate the acquisition of a broad range of transition metals, such as Cu 2+ , Ni 2+ , Co 2+ , and Zn 2+ 16‐19 . Studies have revealed that the metallophore‐based system, but not the AdcABC or ZnuABC system, enables S. aureus or P. aeruginosa to compete with calprotectin for Zn 2+ within hosts and serves as the major Zn 2+ uptake system during infection 10,20‐21 .…”

Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

“…Radiolabeled yNA and pseudopaline are first produced with the reaction of the purified enzymes CntL (for yNA) or a mixture of CntL and CntM (for pseudopaline) with their substrates and then incubated with cell lysates. For the pseudopaline production reaction, we used CntL from P. aeruginosa (PaCntL) or Yersinia pestis (YpCntL) and CntM from Paenibacillus mucilaginosus (PmCntM) that have been shown to be more stable and effective than their equivalent from Pseudomonas aeruginosa (Laffont et al ., 2019). Cloning, expression, and purification of PmCntM have already been described (Laffont et al ., 2019).…”

“…For the pseudopaline production reaction, we used CntL from P. aeruginosa (PaCntL) or Yersinia pestis (YpCntL) and CntM from Paenibacillus mucilaginosus (PmCntM) that have been shown to be more stable and effective than their equivalent from Pseudomonas aeruginosa (Laffont et al ., 2019). Cloning, expression, and purification of PmCntM have already been described (Laffont et al ., 2019). For YpCntL, the gene encoding the YpCntL protein was cloned in a pET‐TEV plasmid (synthetic DNA from GenScript).…”

“…To date, our knowledge of the metallophore‐mediated metal uptake capabilities of P. aeruginosa has been limited to two iron‐specific siderophores, pyochelin, and pyoverdine (Schalk and Cunrath, 2016) and a recently discovered broad‐spectrum metallophore named pseudopaline (Gi et al ., 2015; Lhospice et al ., 2017; Mastropasqua et al ., 2017; McFarlane and Lamb, 2017). Pseudopaline is distantly related to plant nicotianamine and belongs to the same opine‐type broad‐spectrum metallophore family as staphylopine and yersinopine synthetized by Staphylococcus aureus and Yersinia pestis, respectively (Ghssein et al ., 2016; McFarlane et al ., 2018; Laffont et al ., 2019). The cntOLMI genes of P. aeruginosa encode for the proteins involved in pseudopaline biosynthesis and transport (Lhospice et al ., 2017).…”